Biology subjects

Alexandrescu, A. T.

Publications and source records attributed to Alexandrescu, A. T..

6 recordsLinked to original sources

bioRxiv2025

Solution Structure of the Novel CH-domain zinc finger from the puberty regulator Makorin-3

The makorin (MKRN) family of E3 ubiquitin ligases (MKRN1-4) regulates diverse biological processes, including reproduction, neurogenesis, and immune function. The first identified member, MKRN3, is an inhibitor of sexual development that is the site of inherited mutations linked to central precocious puberty (CPP). All makorin proteins share a distinctive cysteine/histidine-rich (CH) domain that has not been previously characterized experimentally. In MKRN3, the CH domain lies between the second C3H zinc finger and the RING domain. The C3H(2)-CH-RING segment appears particularly sensitive to CPP mutations suggesting it may constitute a structure-function unit. Using CD and NMR spectroscopy, we show that the CH domain folds upon coordination of a single Zn2+ ion with picomolar affinity. Spectroscopic and NMR pH-titration analyses identify a CCHC-type metal-binding site, typical of zinc fingers with protein-interaction functions. The NMR structure reveals the CH-domain adopts a canonical {beta}{beta} zinc finger fold, despite atypical ligand spacing and the absence of conserved hydrophobic residues that usually stabilize this type of motif. Thermal denaturation monitored by multiple spectroscopic probes indicates sequential unfolding, with side-chain packing disrupted near 33 {degrees}C but zinc-stabilized secondary structure persisting to [~]63 {degrees}C, consistent with a molten-globule intermediate at high temperature. The function of the CH-domain remains unknown, but it could play a role in allosterically transmitting information on the RNA-bound state of the preceding C3H(2) domain to the subsequent RING domain. Based on a similar metal ligand spacing to a zinc finger from the protein FAAP20 and AlphaFold modeling, the CH-domain may have a ubiquitin-binding function, but this will need to be verified experimentally as AlphaFold also confidently predicts complexes with unrelated random proteins.

biophysics↗

bioRxiv2025

Zinc-induced folding and solution structure of the eponymous novel zinc finger from the ZC4H2 protein

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil, and a single novel zinc finger with four cysteines and two histidines from which the protein gets its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using a synthetic peptide fragment that the zinc finger of ZC4H2 is genuine, and folds around zinc ion with picomolar affinity. NMR pH titration of histidines and UV-Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger likely perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative unprecedented functions.

biophysics↗

bioRxiv2024

Solution structure of the Z0 domain from transcription repressor BCL11A sheds light on the sequence properties of protein-binding zinc-fingers

The transcription repressor BCL11A, which governs the switch from fetal to adult hemoglobin during development, is the target of the first FDA-approved CRISPR/Cas9 gene-editing therapy in humans. By targeting BCL11A, fetal hemoglobin expression is de-repressed to substitute for defective adult hemoglobin in inherited diseases including beta-thalassemia and sickle-cell anemia. BCL11A has six CCHH-type zinc-fingers of which domains 4-6 are necessary and sufficient for dsDNA binding. Here, we focus on the CCHC-type ZNF at the N-terminus of BCL11A (residues 46-72), Z0, thought to modulate oligomerization of the transcription repressor. Using NMR and CD spectroscopy, Z0 is shown to be a thermostable CCHC zinc-finger with a pM dissociation constant for zinc. The NMR structure of Z0 has a prototypical beta-beta-alpha fold, with a hydrophobic knob comprising about half the structure. The unusual proportion of hydrophobic residues in Z0 led us to investigate if this is more general in zinc-fingers that do not bind dsDNA. We used the ZF and WebLogo servers to examine sequences of zinc fingers with demonstrated DNA-binding function, non-binders, and the CCHC-type family of protein-binders. DNA-binders are distinguished by contiguous stretches of high-scoring zinc-fingers. Non-DNA-binders show a depletion of polar residues at the positions expected to contact nucleotides and increased divergence from sequence consensus making these domains more likely to be annotated as atypical, degenerate, or to be missed as zinc-fingers. We anticipate these sequence patterns will help distinguish DNA-binders from non-binders, an open problem in the functional understanding of zinc-finger motifs.

biophysics↗

bioRxiv2024

Templated trimerization of the phage L decoration protein on capsids

The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec1-98) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.

biochemistry↗

bioRxiv2024

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a {Delta}taz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a {Delta}crd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and -ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

biophysics↗

bioRxiv2023

NMR structure verifies the eponymous degenerate zinc finger domain of transcription factor ZNF750

ZNF750 is a nuclear transcription factor that activates skin differentiation and has tumor suppressor roles in several cancers. Unusually, ZNF750 has only a single zinc-finger (ZNF) domain, Z*, with an amino acid sequence that differs markedly from the CCHH family consensus. Because of its sequence differences Z* is classified as degenerate, presumed to have lost the ability to bind the zinc ion required for folding. AlphaFold predicts an irregular structure for Z* with low confidence. Low confidence predictions are often inferred to be intrinsically disordered regions of proteins, which would be the case if Z* did not bind Zn2+. We use NMR and CD spectroscopy to show that a 25-51 segment of ZNF750, corresponding to the Z* domain, folds into a well-defined antiparallel {beta}{beta} tertiary structure with a pM dissociation constant for Zn2+, and a thermal stability >80 {degrees}C. Of three alternative Zn2+ ligand sets, Z* uses a CCHC rather than the expected CCHH motif. The switch in the last ligand maintains the folding topology and hydrophobic core of the classical ZNF motif. CCHC ZNFs are associated with protein-protein interactions but Z* binds DNA. Since the metal chelating site is on the other side of the molecule, it suggests functional preferences are a result of divergent evolution rather than physical constraints on the structure. The structure of Z* provides a context for understanding the domains DNA-binding properties and mutations associated with cancers. We expect other ZNFs currently classified as degenerate, are CCHC-type structures like Z*.

molecular biology↗